Our previous work has revealed that a novel small viral RNA (sRNA) has an essential role in bacteriophage phi29 DNA packaging. The sRNA is co- purified with the prohead but it is not found in the mature virion. Recently, involvement of RNA in DNA packaging has been demonstrated in phages lambda and D108, a Mmu-like phage. Similar to phi29, small RNAs without protein encoding capacity have also been found to be present in orthopoxvirus infected cells. If the involvement of sRNA in viral DNA packaging is a general phenomenon, then these sRNA molecules could be targets for anti-viral therapy. A major objective of this study is to elucidate fundamental mechanisms of the sRNA-protein interaction and the sRNA-DNA interaction during the maturation of the virus in order to understand the role of the sRNA in DNA packaging. Experiments will include: 1) isolation of phi29 DNA packaging intermediates; 2) analysis of the primary structure of the sRNA; 3) comparison of the secondary structure of the prohead-associated sRNA to that of the purified unassociated sRNA; 4) identification of sites for sRNA-DNA and sRNA-protein interactions; 5) determination of the active domain(s) of the sRNA; 6) calculation of the number of sRNA molecules required to package one DNA molecule; 7) studies on the rosette decamers resulting from sRNA-portal vertex interaction, which might provide information to support our model of the role of the sRNA in phi29 DNA packaging, and 8) studies on the antisense RNA against viral infections.